| 摘要: | 為了提升C端硬脂基化Indolicidin (ILs) 的轉染效率，我們將輔助脂質二油酰磷脂酰乙醇胺(DOPE)與ILs以莫耳比為1:3混合 (ILs0.3L)，由先前實驗結果已知ILs0.3L之轉染效果高於ILs。為了瞭解其機制，我們進行了一連串的分析。由圓二色(CD)光譜得知，改質前的IL完全無α螺旋(α-helix)結構，ILs與ILs0.3L則於酸鹼環境皆有α-helix結構，表示IL在添加硬脂基後，有自組裝結構形成，因而導致二級結構改變。利用小角度X-ray散射(SAXS)、動態光散射(DLS)與穿透式電子顯微鏡(TEM)進行結構之構型及粒徑分析，在中性環境，ILs為柱狀微胞結構，ILs0.3L為單層囊泡結構，且ILs所形成的整體結構粒徑較大；而添加DNA後，發現ILs0.3L /DNA粒徑較ILs/DNA小，較易進入細胞。流式細胞儀實驗證實ILs0.3L與ILs皆以內吞作用將DNA帶入細胞，且ILs0.3L載體較ILs組多出巨胞飲作用進入細胞。在酸性環境下ILs0.3L/DNA則有結構重組以及零碎的結構散出的現象，且由肝素競爭實驗可知， 因酸性環境時的結構不穩定，易與DNA分離，這應有助於基因藉由ILs0.3L攝入後的蛋白質表現。共軛焦顯微鏡實驗則顯示ILs0.3L與ILs皆有良好的內體逃脫能力。綜合上述結果，ILs0.3L因具有較小的粒徑、較多進入細胞的途徑、可由內體逃脫以及容易與DNA於胞內脫離等特性，使其傳輸能力高於ILs。;To improve the transfection efficiency of C-terminal stearyl Indolicidin (ILs), a helper lipid, dioleoylphosphatidylethanolamine (DOPE), has been applied to mix with ILs at a molar ratio of 1:3 (ILs0.3L). Previous study indicates that ILs0.3L exhibits higher transfection efficiency than that of ILs. Therefore, a series of analyses were perfomed in this study to understand the promotion mechanism of helper lipids. The circular dichroism (CD) spectra showed that the structure of IL was completely random coiled, whereas ILs and ILs0.3L both exhibited partial α-helix properties. These results indicated that stearyl modiciation allow IL to self-assemble, which also led secondary structure formation. We used small angle X-ray scattering (SAXS), dynamic light scattering (DLS), and transmission electron microscope (TEM) to analyze self-assembled structures and their sizes. In a neutral environment, ILs and ILs0.3L formed cylindrical micelles and unilamellar vesicles, respectively. The overall structure of ILs0.3L was smaller than ILs. ILs0.3L/DNA was also smaller than ILs/DNA, suggesting that ILs0.3L/DNA may enter cells easier. The flow cytometry experiments showed that DNA delivered by ILs0.3L and ILs both through endocytosis. Interestingly, only ILs0.3L/DNA could enter cells through macropinocytosis. In an acidic environment, TEM results showed that the structure of ILs/DNA became compact, whereas ILs0.3L/DNA demonstrated structural reorganization and loose structures. Due to the unstability of ILs0.3L/DNA, the heparin competition experiment showed that acidic environment promoted DNA separated from ILs0.3L, which is essential for the protein expression of ingested genes. Confocal microscopy also showed that DNA delivered by ILs0.3L and ILs could both escape from endosome. Based on the abovementioned results, ILs0.3L owns smaller particle size, additional access to cells, and the capabilities of endosomal escape as well as intracellular DNA release, so its transfection efficiency is better than that of ILs. |
